The goal of this project is determination of the structure of the metal binding sites that form the inducer binding domains of transcriptional repressors that regulate plasmid-encoded bacterial resistances. In common with many drug and antibiotic resistances, the arsenical (ars) and cadmium (cad) resistance operons encode transport ATPases for the extrusion of As(III)/Sb(III) or Pb(II)/Cd(II)/Zn(II). The ArsR and CadC repressors are two small homologous metalloregulatory proteins responsible for metal-regulated gene expression of the ars and cad operons. Residues required for each inducer binding domain will be determined. In addition, in ars operons there is a second As(III)/Sb(III)-responsive repressor, ArsD, which does not exhibit homology to any known metal binding protein. ArsR and ArsD form a regulatory circuit that senses both low and high concentrations of environmental metalloid. The residues involved in As(III)/Sb(III) binding to ArsD will be determined. Finally, the copper (copA) resistance gene encodes a Cu(I)-translocating P-type ATPase. Expression of copA is regulated by copper or silver. The putative CopR regulatory protein will be identified and characterized. Our studies will define new classes of metal binding motifs. This may be more general applicability, since nearly all transport-related drug resistances are transcriptionally regulated, and the drug binding motifs of the regulatory proteins are largely unknown. Thus the repressors of the genes for these efflux pumps provide good model systems for the study of regulation of transmissible bacterial antibiotic resistances.